Angiotensin and thyroxine binding globulin (TBG) are two physiologically critical human plasma precursor decapeptide angiotensin I. It is therefore critical for blood pressure regulation. TBG is the principal and highest affinity binding and transport protein for the thyroid hormones thyroxine (TM) and triiodothyronine (T3). Neither is known to have any ability to act as proteinase inhibitor, yet both are members of the serpin superfamily. We consider that it is not a coincidence that each is a serpin that there has been co-evolution. of the more obvious biological function with less obvious that each is a serpin and that there has been co- evolution of the more obvious biological function with less obvious auxiliary properties that rely on the presence of the serpin fold. We have developed two provocative and novel hypotheses that will be tested in this project. Concerning angiotensinogen, we propose that formation of higher molecular weight complexes of angiotensinogen, either with itself through a non-covalent serpin-based polymerization mechanism, or with pro-eosinophil granule major basic protein (proMBP), serves to alter the accessibility of renin to its target cleavage site in the N-terminus of angiotensinogen and thereby regulates the production of angiotensin peptides. Concerning TBG, we propose that the reactive center loop has been optimized to give substrate-like cleavage by proteinase, with consequent intramolecular serpin-like conformational change, that weakens the affinity for T3 and T4 and thereby provides a proteinase- regulated mechanism for hormone release. The corollary in both cases is that mutations in the serpin core that affect these conformational changes will be detrimental. Three specific aims will test these hypotheses: Aim 1 will use spectroscopic and kinetic methods to determine whether the N- terminus of angiotensinogen is mobile, but is made less accessible to renin by formation of high molecular weight complexes. Aim 2 will examine whether angiotensinogen is a metastable serpin designed to form loop-sheet oligomers, but to be incapable of self-loop insertion on cleavage and will examine the properties of variants, M235T and T174M, correlated with hypertension. Aim 3 will characterize the binding of thyroid hormones to TBG in native and cleaved states, to determine whether loop insertion on cleavage, but without proteinase inhibition, is critical for TBG function, and to examine the properties of TBG oligomers and latent TBG to see how they further understanding of the dysfunction of natural variants.